Signals from the environment or from other cells activate proteins called transcription factors. These proteins bind to regulatory regions of a gene and increase or decrease the level of transcription. By controlling the level of transcription, this process can determine when and how much protein product is made by a gene.
Other chapters in Help Me Understand Genetics. Genetics Home Reference has merged with MedlinePlus. Learn more. The information on this site should not be used as a substitute for professional medical care or advice. As reported previously, there is a large variability in the number, orientation, spacing, and sequences of PREs among all promoters Chou et al , ; Su et al , Despite these similarities, we have observed drastic differences in their expression kinetics.
Therefore, we decided to use p AGA1 and p FIG1 as model promoters of their categories and decipher their mode of regulation. The sequences of each binding sites are detailed above, with capital nucleotides matching the consensus sequences and small nucleotides being mutations from the consensus. The numbers between sites represent the distance in bp between them or the ATG.
Blue arrows represent nucleosomes position Brogaard et al , Each data point is the mean of three biological replicates, and the error bars represent their standard deviation.
However, the most interesting knockouts are the ones that perturbed one promoter to a greater extent than the other one group III, Fig EV4. Only a small percentage of cells induce p FIG1.
Kar4 has been identified as a transcription factor required for the induction of genes implicated in karyogamy, a late event of the mating Kurihara et al , It has also been suggested that Kar4 forms a heterodimer with Ste12, and therefore, the association of those two proteins on the promoter allows the transcription of the late genes Lahav et al , Moreover, we found that KAR4 is induced as early as p AGA1 during the mating response, making it a good candidate to regulate late genes.
Lines represent the median of either the mutant red or the WT strain black for one representative experiment, with the solid line representing the median and the shaded area representing the 25 th —75 th percentile.
Representative microscopy images of the indicated mutant at the specified time point of the experiment. Arrows indicate shmooing events. Scale bars represent 2. We noticed that the presence of these tags slightly influences the dynamics of transcription although the differential response of the two promoters is maintained Appendix Fig S15A and B. Ste12 and Kar4 reach a maximal accumulation at 30 min after the stimulus, a time point where chromatin remodeling starts to take place.
As a consequence, the resulting mRNA production is delayed at this locus. The ability of TFs to bind promoter regions is known to depend on the positioning of nucleosomes on the DNA. This conformation would allow the formation of a Ste12 dimer under basal conditions. The Ste12 dimer on p AGA1 could allow a fast induction of transcription as soon as Fus3 activity is present to derepress Dig1 and Dig2.
In order to understand the parameters that control the dynamics of induction of the late promoters, we performed a series of mutations to test whether we succeeded to accelerate the dynamics of induction of the p FIG1 promoter. This promoter variant was only marginally faster than the WT promoter.
It is faster and more expressed than the natural p FIG1 promoter and retains a Kar4 dependency. Bringing this more labile nucleosome on the p FIG1 promoter accelerates the expression of this construct. However, p BAR1 displays a high level of basal expression; therefore, the identified nucleosome on this promoter must be loosely bound probably allowing Ste12 binding to its target site.
More detailed measurement should be performed to assess whether Ste12 can access this site under basal conditions. Interestingly, Kar4 has been found associated with the AGA1 promoter in basal condition, but its deletion does not alter the level of expression or the dynamics of induction of this early promoter. Therefore, our data demonstrate a more global effect of Kar4 on mating genes induction than previously thought.
We also observed an interaction between Ste12 and Kar4 that is strongly enhanced by pheromone treatment Fig 3 F. Kar4 presence could stabilize the TFs complex on the promoter allowing a recruitment of the chromatin remodelers, so as to evict the nucleosomes and induce an efficient transcription of the downstream ORF. The delay observed in the late gene expression is thus a combination of the requirement for Kar4 to be transcribed at sufficient levels to allow interaction with Ste12 and slow chromatin remodeling on these loci.
The solid line is the median, and the shaded area represents the 25 th —75 th percentiles of the population. We next wanted to verify whether similar dynamics of gene expression occurred under the physiological conditions of mating. Strikingly, under these conditions, we also observed a clear difference in the activation of the two reporters. The single cell traces of of these events recorded in one experiment were aligned temporally to their fusion time, set to 0.
These quantifications reveal very clearly that the induction of p AGA1 gradually increases until it reaches a peak prior to fusion Fig 4 B. In comparison, the FIG1 promoter is not active until roughly 30 min before fusion. In addition, these new findings indicate that p FIG1 induction seems to be tightly correlated with the fusion time, while p AGA1 is expressed earlier and with a larger variability Fig 4 C.
It can be sometimes observed in cells in the close vicinity of a set of engaged mating partners Appendix Fig S Fusion events are marked by a white arrow. Activation dynamics of various promoters prior to fusion as measured by dPSTR R in different mating mixtures. Data information: In B and D , the solid line is the median and the shaded area represents the 25 th —75 th percentiles of the population.
We verified that this difference in dynamics of expression is also present for other promoters Fig 4 D and E, Appendix Fig S In agreement with our classification based on exogenous stimulations experiments, early genes are the first ones to be induced in the mating process, followed closely by intermediate genes. Late genes induction precedes the fusion time by only 30 min, a time when cells seem committed to this process.
These experiments provide a better understanding of the key steps in the mating process. As soon as mating pairs are in proximity, the low level of pheromone constantly produced by the cells is sufficient to trigger a low activation of the mating pathway and induction of the expression of early mating genes.
If both partners are able to arrest in G1, they will extend a mating projection toward each other and polarize their sensing and secretory machinery. This will lead to a local increase in pheromone concentration that will be associated with an increase in signal transduction Appendix Fig S20 ; Conlon et al , The exogenous stimulation experiments have demonstrated that a step increase in pheromone concentration leads to a delayed expression of the late genes.
However, in the mating process, the tight synchronization between the increase in MAPK activity, late gene expression, and fusion suggests that the early genes expressed during the sensing phase allow for a precise induction of the late genes when cells detect a further increase in mating pheromone. Taken together, our results demonstrate that yeast cells use a temporal gradient of pheromone to orchestrate the timing of expression of mating genes.
This behavior bears many similarities with morphogen sensing in development. A key question is how this temporal information is encoded to deliver the proper gene expression profile. In the simple settings offered by budding yeast, our data show that both the affinity of the TF binding sites and chromatin state at the promoter determine the concentration threshold and the timing of gene expression. Yeast strains and plasmids used in this study are listed in Appendix Tables S1 and S2.
All constructs were verified by digestion and sequencing. The integrated promoter variant was amplified from genomic DNA and sequenced for confirmation. We also verified that the presence of the cloning sites Apa I and Cla I was not altering the induction of the two promoters data not shown. They were diluted to an OD of 0. Before the experiments, the cells were diluted to an OD of 0. Imaging was started 30 min later, so as to let the cells settle to the bottom to the well.
Briefly, the nuclei of the cells were segmented by thresholding of the CFP images. The cytoplasm object was obtained by removing the nucleus object expanded by two pixels from the cell object. Dedicated scripts in Matlab The Mathworks were written to further analyze the data.
Only cells tracked from the beginning to the end of the movie were taken into consideration. More information can be found on the Life Technologies website. This post is sponsored by Life Technologies. Previous Question: Is it ok to use antibiotics during transfection? Can you give me some advice on how the most effective way to do this.
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